The overall causes of obesity are a sedentary lifestyle and excess energy intake, also known as overnutrition. However, the molecular mechanisms by which overnutrition generates obesity are not fully understood. We hypothesize that overnutrition causes mitochondrial dysfunction by leading to inhibitory protein acetylation. Nicotinamide riboside (NR) is a precursor of NAD and supplementation with NR has been shown to reduce weight gain and metabolic dysfunction observed in a mouse model of overnutrition. Analysis of the mechanisms by which NR prevents weight gain may elucidate the molecular mechanisms that cause obesity. This proposal is linked to a parent study of NR supplementation in the context of overnutrition which includes both a mouse model and a randomized, placebo-controlled pilot study in human adults. The parent study will use mechanistic methods to evaluate the impact of NR supplementation on NAD metabolism in mice given overnutrition. In the human pilot study, the parent study will test the hypothesis that NR supplementation will prevent weight gain in humans in the context of overnutrition. The objective of this K01 proposal is to evaluate alterations in the NAD- targeted and global metabolome that accompany nicotinamide riboside (NR) supplementation in both mice and humans and correlate these metabolomic changes with outcomes (e.g., prevention of weight gain) in both the mouse mechanistic studies and the human clinical trial. The use of metabolomics in both the mouse and human study provides a unique opportunity to identify novel biomarkers of weight gain and NR supplementation and to link these biomarkers with clinically significant changes in NAD metabolism. The first aim is to evaluate the impact of NR supplementation on the comprehensive NAD metabolome in accessible biological samples. We hypothesize that the NAD metabolome will demonstrate changes associated with increased NAD utilization by SIRT3, a mitochondrial protein deacetylase. We will use NAD-targeted metabolomic analyses of urine, blood and feces to evaluate alterations in NAD metabolism associated with NR supplementation. The second aim will explore the impact of NR supplementation in a nontargeted metabolomic analysis. We hypothesize that NR may prevent weight gain by altering metabolic pathways involved in energy expenditure. We will use nontargeted metabolomic analyses and identify important metabolomic profile changes with single value decomposition-based approaches. The results of this project will shed light on the underlying molecular mechanisms that cause obesity and may identify mechanisms for preventing obesity in the context of overnutrition.